Method of shielding biosynthesis reactions from the ambient environment on an array

ABSTRACT

A method of fabricating an array of biopolymers provides a shield for biochemical reactions and biochemical reactants and is particularly useful for those reactions and reactants that are susceptible to reaction with a component of the ambient environment during the fabrication of the array. The method is applicable to the conventional fabrication and synthesis methods used to fabricate a biopolymer array, such as in situ synthesis of biopolymers on an array and the attachment of pre-synthesized biopolymers on to an array. The method comprises applying a non-miscible fluid (NMF) to the array surface where the biopolymers are being synthesized or attached. The NMF is inert and insoluble with the biochemical reactants and other ancillary materials in solution used in conventional synthesis or attachment of biopolymers. The NMF provides a shield between the ambient atmosphere and the biopolymer synthesis materials or the deprotected pre-synthesized biopolymer at the surface of the array during the synthesis or attachment processes. The NMF may be applied as droplets over each feature location on the surface or may be applied by flooding the surface of the array to fully cover the features. Biomonomer or biopolymer solutions are deposited into or through the NMF to the feature locations on the surface of the array where the synthesis or attachment reactions are to take place using conventional deposition equipment to eject the solutions into the NMF. The NMF provides a shield for activated biomonomers that are susceptible to reaction with a component in the ambient environment, such as moisture in the air. Moreover, the NMF provides a shield for pre-synthesized biopolymers that are susceptible to evaporation when deprotected for attachment to the array surface. The method provides a means by which the potential reactivity of the activated biomonomer or deprotected biopolymer with an ambient atmosphere component can be kept low. As a result, biopolymer arrays can be more accurately fabricated.

TECHNICAL FIELD

This invention relates to arrays of biopolymers used in diagnostics,screening, gene expression analysis, and other applications. Inparticular, the invention relates to a method of fabricating biopolymerarrays, such as DNA arrays.

BACKGROUND ART

Polynucleotide arrays, such as DNA or RNA arrays, are known and areused, for example, as diagnostic or screening tools. Such arrayscomprise a plurality of different polynucleotide probes arranged in apredetermined configuration on a substrate. The polynucleotides of theplurality differ by having a different nucleotide sequence. Differentpolynucleotide probes are located at different regions (also known asfeatures or spots) on the substrate, wherein in each region, multiplecopies of the same polynucleotide are usually present.

The array is exposed to a sample of biological material to be evaluated,also known as the “target”. Upon exposure to the target sample, thearray will exhibit a binding pattern, wherein complementary targetpolynucleotides will hybridize or bind to the array polynucleotideprobes during an assay. This binding pattern can be observed, forexample, by labeling all polynucleotide targets (for example, DNA) inthe sample with a suitable label (such as a fluorescent compound), andaccurately observing the fluorescence pattern on the array. Assumingthat the different sequence polynucleotide probes were correctlydeposited in accordance with the predetermined configuration, then theobserved binding pattern will be indicative of the presence and/orconcentration of one or more polynucleotide components of the targetsample.

Biopolymer arrays can be fabricated using either methods of depositionof intact biopolymer species or using in situ synthesis methods. Thedeposition methods basically involve depositing intact biopolymers atpredetermined locations on a substrate that are suitably activated suchthat the intact biopolymers can link thereto. The intact species ofbiopolymers, each having different monomer sequences, may be depositedat different regions of the substrate to yield the completed arrayhaving a predetermined configuration. Typical procedures known in theart for deposition of intact polynucleotide species, particularly DNAsuch as whole oligomers or cDNA, are to load a small volume of DNA insolution in one or more drop dispensers such as the tip of a pin or inan open capillary and, touch the pin or capillary to the surface of thesubstrate. Such a procedure is described in U.S. Pat. No. 5,807,522.When the fluid touches the substrate surface, some of the fluid istransferred from the pin or capillary to the substrate location. The pinor capillary must be washed prior to picking up the next type of DNA forspotting onto the array. This process is repeated for the plurality ofdifferent polynucleotides and, eventually, the desired array having apredetermined configuration is formed. Alternatively, the DNA can beloaded into a drop dispenser in the form of an inkjet head and firedonto the substrate. Such a technique has been described, for example, inPCT publications WO 95/25116 and WO 98/41531, and elsewhere.

The in situ synthesis methods include those described in U.S. Pat. No.5,449,754 for synthesizing peptide arrays, as well as WO 98/41531 andthe references cited therein for synthesizing polynucleotides(specifically, DNA) using phosphoramidite or other chemistry. Such insitu synthesis methods can be basically regarded as iterative steps ofdepositing droplets of. (a) a protected monomer onto predeterminedlocations on a substrate to link with either a suitably activatedsubstrate surface (or with a previously deposited deprotected monomer);(b) deprotecting the deposited monomer so that it can now react with asubsequently deposited protected monomer; and (c) depositing anotherprotected monomer for linking. Different monomers may be deposited atdifferent regions on the substrate during any one cycle so that thedifferent regions of the completed array will carry the plurality ofdifferent biopolymer sequences as desired in the completed array. Insitu synthesis methods may require one or more intermediate furthersteps in each iteration, such as oxidation and washing steps, as arewell known in the art.

In order for an assay to yield accurate results, it is important thatthe different biopolymer features actually be present on the array, thatthey are put down accurately in the desired or predetermined pattern,that the biopolymers are of the correct size, and that each differentfeature be uniformly populated with the respective biopolymer.

In polynucleotide arrays, the conventional in situ synthesis methods usephosphoramidite nucleoside monomers. In order for the phosphoramiditegroup to link to a hydroxyl of a previously deposited deprotectedpolynucleotide monomer, it must first be activated usually by using aweak acid, such as tetrazole. However, an activated phosphoramidite ishighly reactive with moisture in the air. Therefore, unless someprecaution is taken, the activated phosphoramidite can be used up beforethe desired reaction is complete. As a result there is a reduction inthe deposited phosphoramidite monomer available for forming the completepolynucleotide. This problem is present even when the synthesis isperformed in a nitrogen chamber.

Further, the size (volume) of the synthesis droplet on the substratesurface could be very small, such as a few pico- or nano-liters, suchthat the ratio of surface to volume is very high. A high surface tovolume ratio favors the diffusion of moisture into the droplets.Initially, the moisture from the air tends to be adsorbed at the surfaceof the synthesis droplet. Therefore, the phosphoramidite concentrationat the surface of the droplet will tend to be lowest. Consequently, theconcentration of a completed probe polynucleotide at a feature on thearray tends to decrease from the center of a feature toward itsperimeter. Variations in completed probe concentration within a featureresult in a decrease in the concentration of target sample thatconsequently hybridizes to the respective polynucleotide probe.Therefore, the total signal that should be available from the hybridizedtarget is diminished at the particular feature location during opticalevaluation of the array. Further, it should be noted that the watervapor concentration in the ambient atmosphere might vary. Therefore, thesignal from the hybridized target may also vary from array to array,leading to inconsistency in absolute signal generated from differentarrays of a batch when the same concentration of a target isencountered.

The foregoing problems exist particularly where the phosphoramidite ismixed with the activator and the mixture is deposited as a droplet onthe substrate, and even where the activator is deposited onto apreviously deposited droplet containing the phosphoramidite, both assuch are described in PCT publication WO 98-41531. In either case,ambient moisture presents a problem. Furthermore, when one droplet isdeposited on the other, there is no guarantee of efficient mixing suchthat the activated phosphoramidite will be evenly present at thesubstrate surface.

Thus, it would be advantageous to have a means of fabricating biopolymerarrays that lessens the likelihood of deleterious environmentalinfluences on the accuracy of the fabrication. In particular, it wouldbe desirable, in the fabrication of arrays of biopolymers usingbiomonomers with a linking group that must be activated (such as aphosphoramidite), to provide a means by which the potential reactivityof the activated biomonomer with an ambient atmosphere component (suchas water vapor in air) can be kept low.

SUMMARY OF THE INVENTION

The present invention provides a method of fabricating an array ofbiopolymers on a substrate. The method is useful for shieldingbiosynthesis reactions and the bio-reactants from the ambientenvironment. Moreover, the method is useful for shieldingpre-synthesized biopolymers during their attachment to an arraysubstrate. In particular, the method is useful for shieldingbiosynthesis reactions and reaction components during the synthesisprocess or linking process that are susceptible to reaction with acomponent of the ambient environment, for example moisture in the air.The method of the invention is applicable to the conventionalfabrication and synthesis methods used to fabricate a biopolymer array.

In one aspect of the method, the array is fabricated using conventionalin situ techniques. A first biomonomer is deposited onto a substrate forlinking to a surface of the substrate in an array pattern of features byconventional methods. The linked biomonomer is deprotected usingconventional methods, such that the biomonomer can react with subsequentbiomonomers that are added to grow the biopolymer chain. The subsequentbiomonomer may require activation before attachment to the growingchain. The biomonomer and a suitable activation reagent are deposited onthe array for attachment to the deprotected surface-linked biomonomer.The biomonomers are deposited using conventional deposition equipment,such as computer controlled inkjet systems or piezoelectric depositionsystems that are well known in the art. The method of the inventioncomprises applying a non-miscible fluid (NMF) to the array surface wherethe biopolymers are being synthesized. The NMF is inert and insolublewith the ancillary materials, reagents and biomonomers used in thesynthesis of the biopolymers. In accordance with the invention, the NMFprovides a shield between the ambient atmosphere and the biopolymersynthesis materials at the surface of the array during the synthesisprocess. The shield will delay the diffusion of ambient conditions intothe synthesis areas. The subsequent biomonomers are deposited on thearray until a desired biopolymer sequence is synthesized at eachfeature. The NMF may be applied as droplets over each feature locationor may be applied by flooding the surface of the array to fully coverthe features and the growing biopolymer sequences.

The deposition system used to deposit biomonomers in solution on thearray has a head with multiple pulsejets, each of which can dispensefluid droplets onto the substrate. Each such jet includes a chamber withan orifice, and an ejector which, when activated, causes a droplet to beejected from the orifice. According to the invention, the inkjets of thedeposition system are either immersed into the NMF to fire droplets ofthe activated biomonomer through the NMF to the biopolymer synthesissites, or not immersed, but positioned above the level of the NMF tofire the activated biomonomer droplets into the NMF to the biopolymersynthesis sites on the array. In the preferred embodiment, the densityof the NMF is different from the anhydrous solution of solvent,biomonomers and activation reagents.

In a preferred embodiment, the method of fabricating further comprisethe step of deactivating any unreacted activation reagent after theactivated biomonomer is added to the linked biomonomer on the array. Anancillary material that stops the action of the activation reagent(i.e., deactivation reagent) may be added to the array, preferably byflooding the array surface. The deactivation reagent solution has adensity that is different from the density of the NMF to facilitate thedeactivation reagent reaching the unreacted activation reagent atbiopolymer synthesis sites through the NMF shield. The preferredembodiment further comprises the step of removing all the ancillarymaterials and unreacted biomonomer from the array surface so that thegrowing biopolymer chain can undergo other chemistry.

The foregoing steps are repeated, with a biomonomer deposited and linkedto a previously deposited and linked biomonomer on the substrate. Thegrowing biopolymer chain acts as a substrate bound moiety for eachcycle, until all of the biomonomers have been added to the biopolymerarray. In the fabrication of a typical array with multiple features, allof the foregoing steps are repeated at each of multiple differentregions on the same substrate, where it is desired to form thebiopolymer features.

The biopolymers arrays that may be fabricated according to the inventioninclude DNA, RNA, proteins, etc. arrays, for example. Where the array isa polynucleotide or oligonucleotide array (for example, DNA), thebiomonomer is a nucleoside monomeric unit. The activated biomonomer istypically a phosphoramidite according to conventional oligonucleotidesynthesis. Activated phosphoramidites are well known to be highlyreactive with moisture. Without the method of the invention, anactivated phosphoramidite will react with water vapor in ambientatmosphere and be depleted before a sufficient amount of thephosphoramidite has reacted with the growing polynucleotide chains ofthe array, even in a nitrogen chamber.

In another aspect of the invention, a method of fabricating biopolymerarrays from pre-synthesized biopolymers is provided. The pre-synthesizedbiopolymer is deprotected before it is linked to the array surface. Thedeprotected pre-synthesized biopolymer is soluble in aqueous buffersolution. The droplets of the deprotected pre- synthesized biopolymersolution that are deposited for linking to an array substrate are verysmall and have the tendency to evaporate quickly in the ambientenvironment. The method of fabricating according to this embodimentcomprises enclosing the droplets of the deprotected pre-synthesizedbiopolymer solution in the NMF for deposition. The NMF is inert,immiscible and insoluble in aqueous solution. Therefore, the NMF willsurround the droplets and delay the diffusion of the aqueous solutionout of the droplet such that the concentration of the deprotectedpre-synthesized biopolymer will remain relatively constant while itlinks to the surface of the array substrate at each feature.

In still another aspect of the invention, a method of shieldingbiosynthesis reactions and biosynthesis reactants from the ambientenvironment is provided. The method of shielding comprises applying theNMF to one or more sites where the biosynthesis reactions take place.The NMF is inert and insoluble with respect to the biosynthesisreactions and the biosynthesis reactants. The NMF is applied to coverthe biosynthesis site(s). The method of shielding further comprisesdepositing one or more of the sensitive biosynthesis reactants throughthe NMF on the biosynthesis site(s).

In still another aspect of the invention, a shield that protectssensitive biosynthesis reactions and biosynthesis reactants from theambient environment is provided. The shield comprises a non-misciblefluid (NMF) applied to cover the biosynthesis reactions and reactants.The NMF is inert and insoluble with respect to the biosynthesisreactions and the biosynthesis reactants.

The present methods and apparatus provide any one or more of a number ofuseful benefits. For example, in the fabrication of arrays ofbiopolymers using biomonomers with a linking group that must beactivated, the present invention provides a means by which the potentialreactivity of the activated biomonomer with an ambient atmospherecomponent can be kept low. Further, in the fabrication of arrays ofbiopolymers using pre-synthesized biopolymers that are water-solublewhen deprotected for linking to the surface of the substrate, thepresent invention provides a means by which the potential reactivity ofthe deprotected biopolymer in solution with an ambient atmosphere can bekept low.

BRIEF DESCRIPTION OF THE DRAWINGS

The various features and advantages of the present invention may be morereadily understood with reference to the following detailed descriptiontaken in conjunction with the accompanying drawings, where likereference numerals designate like structural elements, and in which:

FIG. 1 illustrates a block diagram of the method of the presentinvention.

FIG. 2 a side view of an apparatus that is used in the method of theinvention according to one embodiment.

FIG. 3 illustrates a side view of an apparatus that is used in themethod of the invention according to another embodiment.

FIG. 4 illustrates a side view of an apparatus that is used in themethod of the invention according to still another embodiment.

FIG. 5 illustrates a side view of an apparatus that is used in themethod of the invention according to yet another embodiment.

FIG. 6a illustrates a side view of an apparatus used in the method ofthe invention according to yet still another embodiment.

FIG. 6b illustrates a magnified view of one portion of the apparatus ofFIG. 6a.

FIG. 7 illustrates a block diagram of another embodiment of the methodof fabricating biopolymer arrays in accordance with the invention.

MODES FOR CARRYING OUT THE INVENTION Definitions

The following terms are intended to have the following general meaningsas they are used herein:

Polynucleotide—a compound or composition that is a polymeric nucleotideor nucleic acid polymer. The polynucleotide may be a natural compound ora synthetic compound. In the context of an assay, the polynucleotide canhave from about 20 to 5,000,000 or more nucleotides. The largerpolynucleotides are generally found in the natural state. In an isolatedstate the polynucleotide can have about 30 to 50,000 or morenucleotides, usually about 100 to 20,000 nucleotides, more frequently500 to 10,000 nucleotides. Thus, it is obvious that isolation of apolynucleotide from the natural state often results in fragmentation.The polynucleotides include nucleic acids, and fragments thereof, fromany source in purified or unpurified form including DNA, double-strandedor single stranded (dsDNA and ssDNA), and RNA, including t-RNA, m-RNA,r-RNA, mitochondrial DNA and RNA, chloroplast DNA and RNA, DNA/RNAhybrids, or mixtures thereof, genes, chromosomes, plasmids, the genomesof biological materials such as microorganisms, e.g. bacteria, yeasts,viruses, viroids, molds, fingi, plants, animals, humans, and the like.The polynucleotide can be only a minor fraction of a complex mixturesuch as a biological sample. Also included are genes, such as hemoglobingene for sickle-cell anemia, cystic fibrosis gene, oncogenes, cDNA, andthe like.

The polynucleotide can be obtained from various biological materials byprocedures well known in the art. The polynucleotide, where appropriate,may be cleaved to obtain a fragment that contains a target nucleotidesequence, for example, by shearing or by treatment with a restrictionendonuclease or other site specific chemical cleavage method.

For purposes of this invention, the polynucleotide, or a cleavedfragment obtained from the polynucleotide, will usually be at leastpartially denatured or single stranded or treated to render it denaturedor single stranded. Such treatments are well known in the art andinclude, for instance, heat or alkali treatment, or enzymatic digestionof one strand. For example, double stranded DNA (dsDNA) can be heated at90-100° C. for a period of about 1 to 10 minutes to produce denaturedmaterial, while RNA produced via transcription from a dsDNA template isalready single stranded.

Oligonucleotide—a polynucleotide, usually single stranded, usually asynthetic polynucleotide but may be a naturally occurringpolynucleotide. The oligonucleotide(s) are usually comprised of asequence of at least 5 nucleotides, usually, 10 to 100 nucleotides, moreusually, 20 to 50 nucleotides, preferably, 10 to 30 nucleotides, morepreferably, 20 to 30 nucleotides, and desirably about 25 nucleotides inlength.

Various techniques can be employed for preparing an oligonucleotide.Such oligonucleotides can be obtained by biological synthesis or bychemical synthesis. For short sequences (up to about 100 nucleotides),chemical synthesis will frequently be more economical as compared to thebiological synthesis. In addition to economy, chemical synthesisprovides a convenient way of incorporating low molecular weightcompounds and/or modified bases during specific synthesis steps.Furthermore, chemical synthesis is very flexible in the choice of lengthand region of target polynucleotides binding sequence. Theoligonucleotide can be synthesized by standard methods such as thoseused in commercial automated nucleic acid synthesizers. Chemicalsynthesis of DNA on a suitably modified glass or resin can result in DNAcovalently attached to the surface. This may offer advantages in washingand sample handling. For longer sequences, standard replication methodsemployed in molecular biology can be used, such as the use of M13 forsingle stranded DNA as described in J. Messing (1983) Methods Enzymol.101:20-78.

Other methods of oligonucleotide synthesis include phosphotriester andphosphodiester methods (Narang, et al. (1979) Meth. Enzymol. 68:90) andsynthesis on a support (Beaucage, et al. (1981) Tetrahedron Letters22:1859-1862) as well as phosphoramidite techniques (Caruthers, M. H.,et al., “Methods in Enzymology,” Vol. 154, pp. 287-314 (1988) and othersdescribed in “Synthesis and Applications of DNA and RNA,” S. A. Narang,editor, Academic Press, New York, 1987, and the references containedtherein. The chemical synthesis via a photolithographic method ofspatially addressable arrays of oligonucleotides bound to glass surfacesis described by A. C. Pease, et al., Proc. Nat. Aca. Sci. USA (1994)91:5022-5026.

For the purposes of this invention, the terms “oligonucleotide” and“polynucleotide” are used interchangeably, unless otherwise noted.

Oligonucleotide or Polynucleotide Probe—an oligonucleotide employed tobind to a portion of a polynucleotide, such as another oligonucleotideor a target nucleotide sequence. The design and preparation of theoligonucleotide probes are generally dependent upon the sensitivity andspecificity required, the sequence of the target polynucleotide and, incertain cases, the biological significance of certain portions of thetarget polynucleotide sequence.

Monomer—A member of the set of small molecules which can be joinedtogether to form a polymer. The set of monomers includes but is notrestricted to, for example, the set of common L-amino acids, the set ofD-amino acids, the set of synthetic amino acids, the set of nucleotidesand modified nucleotides, and the set of pentoses and hexoses. Otherexamples include a basic phosphodiesters, such as polyethers, andprotein-nucleic acid (PNA) hybrids. As used herein, monomers refers toany member of a basis set for synthesis of a polymer. For example,dimers of the 20 naturally occurring L-amino acids form a basis set of400 monomers for the synthesis of polypeptides. Different monomers maybe used at successive steps in the synthesis of a polymer. Furthermore,each of the monomers may include protected members that are modifiedafter synthesis. A monomer may also include modified monomers.

Phosphoramidite—For the purposes of the invention, the term“phosphoramidite(s)” includes phosphite(s) and H-phosphonate(s). FormulaI covers phosphoramidites, phosphites and H-phosphonates:

in which:

A represents H or an optionally protected hydroxyl group;

B is a purine or pyrimidine base whose exocyclic amine functional groupis optionally protected;

C is a conventional protective group for the 5′-OH functional group (forthe purposes of the definition herein the “C” does not represent carbonper se);

x=0 or 1 provided:

a) when x=1:

R₃ represents H and R₄ represents a negatively charged oxygen atom; or

R₃ is an oxygen atom and R₄ represents either an oxygen atom or anoxygen atom carrying a protecting group; and

b) when x=0:

R₃ is an oxygen atom carrying a protecting group and R₄ is either ahydrogen or a di-substituted amine group.

When x is equal to 1, R₃ is an oxygen atom and R₄ is an oxygen atom, thesynthesis method is in this case the so-called phosphodiester method;when R₄ is an oxygen atom carrying a protecting group, the synthesismethod is in this case the so-called phosphotriester method.

When x is equal to 1, R₃ is a hydrogen atom and R₄ is a negativelycharged oxygen atom, the synthesis method is known as the H-phosphonatemethod.

When x is equal to 0, R₃ is an oxygen atom carrying a protecting groupand R₄ is either a halogen, the synthesis method is known as thephosphite method and, when R₄ is a leaving group of the di-substitutedamine type, the synthesis method is known as the phosphoramidite method.

Phosphoramidites and nucleoside phosphoramidites are described in U.S.Pat. No. 5,902,878, U.S. Pat. No. 5,700,919, U.S. Pat. No. 4,415,732,PCT publication WO 98/41531 and the references cited therein, amongothers, all incorporated by reference. A “group” includes bothsubstituted and unsubstituted forms.

Nucleotide—the monomeric unit of nucleic acid polymers, i.e., DNA andRNA, that comprises a nitrogenous heterocyclic base, which is aderivative of either a purine or pyrimidine, a pentose sugar, and aphosphate (or phosphoric acid) and includes modified nucleotides. Whenthe phosphate is removed, the monomeric unit that remains is a“nucleoside”. Thus a nucleotide is a 5′-phosphate of the correspondingnucleoside. When the nitrogenous base is removed from the nucleotide,the monomeric unit that remains is a “phosphodiester”. For the purposesof the invention, “nucleotide” includes its corresponding nucleoside andphosphodiester, and “oligonucleotide” includes its correspondingoligonucleoside and oligophosphodiester, unless indicated otherwise.

Modified Nucleotide—a unit in a nucleic acid polymer that contains amodified base, sugar and/or phosphate group. The modified nucleotide canbe produced by a chemical modification of a nucleotide either as part ofthe nucleic acid polymer or prior to the incorporation of the modifiednucleotide into the nucleic acid polymer. For example, the methodsmentioned above for the synthesis of an oligonucleotide may be employed.In another approach a modified nucleotide can be produced byincorporating a modified nucleoside triphosphate into the polymer chainduring an amplification reaction. Examples of modified nucleotides, byway of illustration and not limitation, include dideoxynucleotides,derivatives or analogs that are biotinylated, amine modified, alkylated,fluorophore-labeled, and the like and also include phosphorothioate,phosphite, ring atom modified derivatives, and so forth.

Biomonomer—a single biological monomer unit, which can be linked withthe same or other biomonomers to form a biopolymer. For example, asingle amino acid or nucleotide with two linking groups, one or both ofwhich may have removable protecting groups, is a biomonomer. Abiomonomer fluid or solution or biopolymer fluid or solution refers to aliquid containing either a biomonomer or biopolymer, respectively,typically in a solution comprising an ancillary material.

Biopolymer—a polymer found in biological systems comprising a pluralityof biological monomeric units or biomonomers linked together, such asnucleic acids (including DNA, RNA, polynucleotides, oligonucleotides,oligonucleotide probes) sugars, proteins, antibodies, antigens, enzymes,coenzymes, ligands, receptors, hormones and labels, and genes thatspecify any of the above. Biopolymers include compounds composed of orcontaining amino acid or nucleotide analogs or non-nucleotide groups.This includes polynucleotides in which the conventional backbone hasbeen replaced with a non-naturally occurring or synthetic backbone, andnucleic acids in which one or more of the conventional bases has beenreplaced with a synthetic base capable of participating in Watson-Cricktype hydrogen bonding interactions.

Substrate or Surface—a porous or non-porous water insoluble material.The surface can have any one of a number of shapes, such as strip,plate, disk, rod, particle, including bead, and the like. The substratecan be hydrophilic or capable of being rendered hydrophilic and includesinorganic powders such as silica, magnesium sulfate, and alumina;natural polymeric materials, particularly cellulosic materials andmaterials derived from cellulose, such as fiber containing papers, e.g.,filter paper, chromatographic paper, etc.; synthetic or modifiednaturally occurring polymers, such as nitrocellulose, cellulose acetate,poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose,polyacrylate, polyethylene, polypropylene, poly (4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl butyrate), etc.; either used by themselves or in conjunctionwith other materials; glass available as Bioglass, ceramics, metals, andthe like. Natural or synthetic assemblies such as liposomes,phospholipid vesicles, and cells can also be employed.

Immobilization of oligonucleotides on a substrate or surface may beaccomplished by well-known techniques, commonly available in theliterature. See, for example, A. C. Pease, et al., Proc. Nat. Acad. Sci.USA, 91:5022-5026 (1994).

Ancillary Materials—materials that are conventionally employed in thefabrication of biopolymer arrays. For example, ancillary materialsinclude, but are not limited to buffers, salts, reagents, and solvents.For example, ancillary materials include activation, deactivation,deprotection, oxidizing, reducing and capping agents or reagents andanhydrous solvents used in biopolymer synthesis or in pre-synthesizedbiopolymer attachment to an array.

The term “alkyl” refers to a monoradical branched or unbranchedsaturated hydrocarbon chain preferably having from 1 to 40 carbon atoms,more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6carbon atoms. This term is exemplified by groups such as methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, n-decyl, tetradecyl,and the like, unless otherwise indicated.

The term “alcohol” refers to an alkyl containing one or more hydroxylgroups.

The term “alkyl alcohol” refers to a substituted alkyl having an alcoholgroup or a substituted alcohol having an alkyl group.

The term “halogen” refers to fluoro, chloro, bromo and iodo groups.

The term “haloalkylalcohol” or “halogenalkylalcohol” refers to an alkylalcohol as defined above substituted by 1 to 4 halo groups as definedabove, which may be the same or different, such as trifluoromethyl-,trichloroethyl-alcohol, and the like.

It will also be appreciated that throughout the present application,words such as “upper”, “lower” and the like are used with reference to aparticular orientation of the apparatus with respect to gravity, but itwill be understood that other operating orientations of the apparatus orany of its components, with respect to gravity, are possible. Referenceto a “droplet” being dispensed from a pulsejet herein, merely refers toa discrete small quantity of fluid (usually less than about 1000 pL)being dispensed upon a single pulse of the pulsejet (corresponding to asingle activation of an ejector) and does not require any particularshape of this discrete quantity. When a “spot” is referred to, this mayreference a dried spot on the substrate resulting from drying of adispensed droplet, or a wet spot on the substrate resulting from adispensed droplet which has not yet dried, depending upon the context.“Fluid” is used herein to reference a liquid. Use of the singular inreference to an item, includes the possibility that there may bemultiple numbers of that item.

DETAILED DESCRIPTION OF THE INVENTION

A block diagram of the method 100 of fabricating an array of biopolymerson a substrate is illustrated in FIG. 1. The method 100 is useful forshielding the reactions and reactants in biopolymer synthesis, such asthe growing biopolymer chain or a biomonomer to be added to the growingchain. In particular, the invention is useful for shielding reactions orreactants that are or become sensitive to a component of the ambientenvironment during the synthesis process. For example, the method isuseful for shielding a biomonomer that, upon activation for attachmentto the biopolymer, becomes susceptible to reaction with moisture in theair, or shielding a pre-synthesized biopolymer that, upon deprotectionfor attachment to an array substrate, becomes susceptible toevaporation.

The method 100 of fabricating an array of biopolymers from biomonomersfollows the conventional in situ synthesis chemistry for the synthesisof each type of biopolymer, such as oligonucleotides, proteins, etc. Anarray substrate is prepared so that a biomonomer can be linked to itssurface using conventional methods. The biomonomer is deposited onto thesurface of the substrate and is suitably linked thereto in an arraypattern of features. According to conventional synthesis steps, thesubstrate-bound biomonomer is then chemically prepared to receive orlink to another biomonomer. Different or the same biomonomers aresubsequently added to the locations on the array features in aniterative process to synthesize the same or different biopolymers. Thenumber of biopolymers and their sequence make-up, depends on theparticular assay to be performed using the array.

The biomonomer may be in solution comprising a solvent or otherancillary materials for deposition. For example, in the synthesis ofoligonucleotides, typically the phosphoramidite is dissolved in ananhydrous solvent for deposition. Conventional anhydrous solvents thatare used in phosphoramidite deposition are acetonitrile, propylenecarbonate, adiponitrile, etc. The biomonomers that are subsequentlyadded are chemically prepared, or may be activated, to link to the arraybound biomonomer. In the synthesis of oligonucleotides, aphosphoramidite is activated by reaction with a reagent, such astetrazole, ethylthiotetrazole, dicyanoimidazole, or benzimidazoliumtriflate, for example. Phosphoramidites and nucleoside phosphoramiditesare well known in the art and described in, for example, U.S. Pat. No.5,902,878, U.S. Pat. No. 5,700,919, U.S. Pat. No. 4,415,732, PCTpublication WO 98/41531 and the references cited therein, among others,all of which are incorporated herein by reference. Therefore, thesubsequent biomonomer solution may also contain an activation reagent oractivator. Other conventional ancillary materials may be present also.

A long-standing problem in in situ biopolymer array synthesis is theeffect of environmental conditions on the synthesis process. Forexample, if one of the reagents or biomonomers is susceptible toreaction with components of the environment, for example, moisture inthe ambient, the moisture will react immediately with the reagent orbiomonomer until total hydrolysis thereof. Therefore, biopolymersynthesis is performed typically in a nitrogen chamber that isessentially devoid of moisture. However, biopolymer synthesis involvesworking with very small quantities in solution, such as droplets of afew pico- or nano-liters of product in solution. The surface-to-volumeratio of these droplets is very high and susceptible to even very smallquantities of moisture. The high surface-to-volume ratio essentiallyfavors the impact of diffusion of moisture into the droplets. Typicalnitrogen chambers do not adequately prevent such small quantities ofmoisture, for example, from interfering with biopolymer synthesis.Therefore, without additional precautions, the droplets of reactivesolution will react with components in the ambient environment, even ina nitrogen chamber, and will be depleted before all of activatedbiomonomer is allowed to react and become a part of the biopolymer.

In accordance with the invention, the method 100 comprises applying 101a non-miscible fluid (NMF) to the array surface where the biopolymersare being synthesized. The NMF completely covers the synthesis area(s)and provides an environmental shield for the synthesis reactants andreactions. According to the invention, the NMF is inert and insolublewith respect to the biomonomers, biopolymers, reagents and otherancillary materials used in the synthesis of the biopolymers. Foroligonucleotide synthesis, the NMF is inert and insoluble with respectto the phosphoramidite biomonomer, its activator reagent and theanhydrous solvent for example.

For the purposes of the invention, the term “non-miscible” has itsconventional definition. The NMF does not mix with the biomonomer,reagents, anhydrous solvents and other ancillary materials used in thesynthesis processes. The term “inert” has its conventional definition.The NMF does not chemically react with the biomonomer, reagents,anhydrous solvent or other ancillary materials. Moreover, the term“insoluble” has its conventional definition. The solubility of the NMFin the solution of biomonomer and activator reagent in the anhydroussolvent is less than about 10 mg per milliliter. In addition, the NMFshould have a low affinity for the environmental component(s) in theambient atmosphere that the NMF is intended to shield against. Forexample, the NMF should have a low affinity for water, such thatmoisture vapor does not penetrate the NMF to reach a biomonomer that issensitive to moisture. The solubility of water in the NMF is below 0.2 gof water per kilogram of NMF at 20° C., and preferably below 0.13 g ofwater per kilogram of NMF at 20° C., and depends on the particular NMF.

Some NMF that are particularly useful for the invention are listed inTable 1. The list in Table 1 is illustrative only. As long as the NMFhas the characteristics described above, it is within the scope of theinvention. In the case of oligonucleotide synthesis, preferably heptaneprovides the characteristics necessary for shielding phosphoramiditesynthesis conditions and materials, from the ambient, but any otherfluid with similar properties to heptane could be used. However, becauseof the melting temperature of heptadecane, the temperature must be keptabove 20° C. when heptadecane is used as the NMF.

The method 100 of the invention further comprises the step of depositing103 the biomonomer solution, which is suitable activated for attachmentto the surface of the array, or to a surface-bound biomonomer on thearray, with conventional deposition equipment, such as computercontrolled inkjet systems or piezoelectric deposition systems that arewell known in the art.

The deposition system used to deposit biomonomers in solution on thearray has a head with multiple pulsejets, each of which can dispensefluid droplets onto the substrate. Each such jet includes a chamber withan orifice, and an ejector which, when activated, causes a droplet to beejected from the orifice. There is typically one pulsejet for eachdifferent biomonomer to be deposited. The ejected droplet has sufficientspeed to penetrate the NMF and reach the surface of the array where thebiopolymer is being synthesized.

According to one embodiment of the method 100, the step of depositing103 comprises the step of immersing the pulsejets into the NMF beforethe step of ejecting droplets of the biomonomer solution. FIG. 2illustrates this embodiment, however only one pulsejet is illustratedfor simplicity. The array substrate 20 comprises an array of biopolymersynthesis sites 22 on the surface 23 thereof. The array 20 is placed inthe bottom (surface 23 side up) of a vessel 24 that is constructed tohold fluids.

TABLE 1 Representative Non-miscible Fluids melting boiling Non-miscibletempera- point Density viscosity solubility with Fluids (NMF) ture (°C.) (° C.) (g/ml) (cP at 20° C.) acetonitrile at 20° C. heptane 98-990.683 0.409 immiscible octane 125-126 0.702 0.542 immiscible nonane150-151 0.718 0.711 immiscible decane 173-174 0.73 0.92 immiscibleundecane 195-196 0.742 1.17 immiscible dodecane 241-216 0.748 1.35immiscible tridecane −5.50 235-236 0.755 1.55 immiscible tetradecane5.0-7.0 250-253 0.762 2.18 immiscible pentadecane  8.0-10.0 269-2700.769 2.81 immiscible hexadecane 17.0-19.0 283-286 0.773 3.34 immiscibleheptadecane 20.0-22.0 303 0.778 immiscible cycloheptane 116-118 0.8121.64(13.5C) immiscible cyclooctane 150-152 0.836 2.35(13.5C) immisciblecyclononane immiscible cyclodecane  9.0-10.0 201 0.858 immiscible

The vessel 24 holds a sufficient quantity of NMF to completely cover thearray substrate 20 and allow a portion of the pulsejet 26 to be immersedtherein. In this embodiment, the NMF is applied (step 101) by floodingthe NMF over the surface 23 of the array 20 within the vessel 24. TheNMF is immiscible with the biopolymer synthesis components and ancillarymaterials on the surface 23 of the array 20. Moreover, preferably theNMF has a different density and a different viscosity than ancillarymaterials used for the biopolymer synthesis, such as the solvents. Morepreferably, the density of the NMF is lower in this embodiment so thatthe biomonomers in solution 28 remain adjacent to the array surface 23and the NMF surrounds and covers the biomonomer solution at thesynthesis sites 22. The pulsejet 26 is immersed into the NMF over thebiosynthesis sites 22 and ejects the biomonomer solution 28 to the site22. The biomonomer solution 28 is shielded by the NMF from theenvironment. The biomonomer solution 28 is suitably activated and linksat a respective synthesis site 22 of a growing biopolymer chain underthe NMF shield. The pulsejet 26 is withdrawn from the NMF (illustratedby the double-headed arrow in FIG. 2), and moved to another synthesissite 22 (which is illustrated by the single headed arrow), where it isreimmersed to eject the next biomonomer solution 28 to the next site 22.

As mentioned above, there is one pulsejet 26 for each different type ofbiomonomer being deposited. For oligonucleotide synthesis, there arefour pulsejets, for example, one for each nucleotide base. In practice,all pulsejets 26 may be immersed into the NMF in the step of immersing.However, not all of the pulsejets 26 will actually fire and eject abiomonomer solution 28 at each step of ejecting. Whether and whichpulsejet 26 actually fires a biomonomer solution 28 depends on thedesired sequence of biomonomers at each synthesis site 22 of the array20 and is controlled by a computer.

FIG. 3 illustrates another embodiment of the method 100, wherein thestep of depositing 103′ comprises the step of immersing the pulse jet(s)26 into the NMF above the surface 23 of the array 20 and ejecting abiomonomer solution droplet 28 to the synthesis sites 22. However, afterthe step of ejecting, the pulsejets are not withdrawn from the NMFbefore moving to the next synthesis site 22, as was described above forFIG. 2. Instead, the pulsejet 26 is just moved through the NMF above thesurface 23 of the array 20 to the next site 22 location. The ejectedbiomonomer droplets 28 reach the surface 23 where they will couple orlink at respective synthesis sites 22 on the array 20.

FIG. 4 illustrates still another embodiment of the method 100, whereinthe step of depositing 103″ comprises the step of ejecting the activatedbiomonomer droplets 28 into the NMF, without first immersing thepulsejets 26 into the NMF, to deposit the biomonomer droplets 28 on thesynthesis sites 22. In this embodiment, the step of depositing 103″ alsocomprises the step moving the pulse jets 26 above the level of the NMF.Since the pulse jets 26 are not immersed in the NMF, the pulsejets 26are not withdrawn from the NMF before they are moved. When thebiomonomer droplets 28 reach the surface 23 of the array 20, they willcouple or link at respective synthesis sites 22 on the array 20.

FIG. 5 illustrates still another embodiment, wherein the orientation ofthe array 20 and the pulsejet 26 is reversed. The array 20 is attachedto a moving arm 30 above the vessel 24′ that is accommodated to hold thepulsejet 26 in the bottom portion thereof. The array 20 surface 23 isflooded with NMF in that the whole surface 23 of the array 20 isimmersed in a quantity of NMF in the vessel 24′. In the step ofdepositing 103′″, the array 20 is immersed in the NMF. The arm 30 movesthe array 20 as the activated biomonomer droplets 28 are ejected fromthe stationary pulse jets 26 through the NMF to respective synthesissites 22, where the biomonomers will couple or link to grow thebiopolymer chain.

FIGS. 6a and 6 b illustrate yet still another embodiment of the method100, wherein the NMF is added (step 101) to the surface 23 of the array20 by depositing individual droplets 31 of NMF that surround or envelopone or more individual biopolymer synthesis sites 22. In thisembodiment, the step of depositing 103″″ comprises the step of ejectingthe biomonomer solution 28 from the pulse jet 26 into the droplet 31 ofNMF. The droplet of biomonomer 28 moves into the NMF droplet 31 to thesurface 23 where the synthesis reaction site 22. FIG. 6b is a magnifiedview of the NMF droplet 31 surrounding the biomonomer droplet 28 thatwas ejected from pulsejet 26 of FIG. 6a. In this embodiment, the NMFdroplet 31 may or may not fully cover or envelop the biomonomer solutiondroplet 28 in order to provide its shielding effects in accordance withthe invention.

In each of the above embodiment, preferably the density of the NMF isdifferent from the density of the biopolymer, biomonomer and ancillarymaterials. More preferably, the density of the NMF in each of theembodiments illustrated in FIG. 2-4, 6 a and 6 b is lower than thedensity of the biopolymer, biomonomer and ancillary materials so thatthe NMF better covers or shields the synthesis site 22 while thebiosynthesis reactions occur on the surface 23. In the embodiment of themethod 100′ illustrated in FIG. 5, the density of the NMF is morepreferably higher than that of the biopolymers, biomonomers andancillary materials to better facilitate the movement and placement ofbiomonomer solution 28 at the synthesis sites 22. For oligonucleotidesynthesis, the NMF has a density higher than acetonitrile, for example.The density of acetonitrile is 0.786 g/ml. Referring to Table 1, a NMFsuch as cyclooctane with a density of 0.836 gram/milliliter would beappropriate to facilitate the movement of the phosphoramidite andactivator in anhydrous acetonitrile solvent droplets 28 to the arraysurface 23 in this embodiment.

As noted above, in each of the embodiments described above, the densityof the activated biomonomer solution 28 is different from the NMF bybeing either higher or lower than the NMF, so that the biomonomersolution 28 effectively travels through the NMF to the synthesis sites22 on the array surface 23 where the droplet remains for the synthesisto take place. In addition to differences in density, the surfacetension of the different fluids facilitates the movement of thebiomonomer droplets 28, as well as the deposition system. The inkjet andpiezoelectric deposition systems can fire the droplets 28 at a highspeed through the NMF shield to reach the surface 23 of the array 20without difficulty. For example, for oligonucleotide synthesis, thefiring speed of the pulsejet 26 is about 10 m/s. Therefore, the affectof having an NMF with a different density from the biomonomer droplets28 is primarily to allow the NMF to surround and shield the activatedbiomonomers at the synthesis sites 22. The anhydrous NMF separates thesynthesis sites 22 and all of the reactants from the atmosphere andshields them temporary from detrimental environmental conditions, suchas moisture. The NMF delays the hydrolysis of reagents with moisture andallows the coupling reaction between biomonomers to be completed beforemoisture reaches the synthesis sites 22.

Referring back to FIG. 1, the method 100, 100′ preferably furthercomprises the step of deactivating or quenching 105 any unreactedbiomonomer or activation reagent after the coupling reaction iscompleted. A solution of a reagent that stops the action of theactivation reagents is added to the array via either another pulse jet26, such that the quenching reagent is fired through the NMF to thesynthesis sites 22, or by flooding the surface of the array 20 with thequenching reagent. Quenching or deactivation reagents are well known inthe art. If the quenching reagent is flooded over the surface of thearray 20, then the quenching reagent preferably has a density that isdifferent from the density of the NMF to displace the NMF or reach theunreacted biomonomer or activation reagent at biopolymer synthesis sites22. For example, some alcohols and water each have densities that arehigher than the NMF. An appropriate alcohol or mixture of an appropriatealcohol and water may be used for deactivation by flooding the surfaceof the array 20. Preferably, the deactivation reagent deactivates theactivation reagents by hydrolysis. For oligonucleotide synthesis, thedeactivation reagent may be methanol, water, alkyl alcohol,halogenalkylalcohol, or trichloroethanol, for example, to hydrolyze thereactants. Moreover, the deactivation reagent solution may have a higherdensity than the NMF to facilitate the reagent reaching the unreactedactivation reagent at biopolymer synthesis sites when fired as dropletsthrough the NMF shield in the embodiments illustrated in FIGS. 2-4, 6 aand 6 b, and may have a lower density than the NMF to facilitate thereagent reaching the unreacted reagents, etc., when fired as dropletsthrough the NMF in the embodiment of FIG. 5. Alternatively, in theembodiment of FIG. 5, the arm 30 which holds and moves the array 20could be withdrawn from the NMF in the vessel 24′ during thedeactivation step, such that the surface 23 of the array and thesynthesis sites 22 are exposed to the moisture in the atmosphere fordeactivation by hydrolysis. In this example, a deactivation reagent isnot needed.

Upon deactivation, the method 100 preferably further comprises the stepof removing 107 all the reagents and solutions from the array surface 23so that the growing biopolymer chains can undergo further synthesischemistry. Typically, one or more of the foregoing steps are repeated(step 109) in an iterative fashion, with a subsequent biomonomer 28being deposited and linked to a previously deposited and linkedbiomonomer on the substrate 20. The growing biopolymer chain acts as asubstrate bound moiety for each cycle, until all of the biomonomers havebeen added to the biopolymer array. In the fabrication of a typicalarray with multiple features, all of the foregoing steps are repeated ateach of multiple different regions 22 on the same substrate 20, where itis desired to form the biopolymer features.

The biopolymers that may be synthesized according to the inventioninclude DNA, RNA, proteins, etc. Where the array is a polynucleotide oroligonucleotide array, the biomonomers are nucleoside monomeric units.Likewise, where the array is a protein array, the biomonomers are aminoacids. Protein arrays can be arrays of antibodies, antigens, ligands andreceptors also, for example. Proteins are synthesized according toconventional methods, such as those described in U.S. Pat. Nos.4,591,570; 5,143,854; and 5,252,743 and the following articles: Ekins,R., et al., “Development of microspot multi-analyte ratiometricimmunoassay using dual fluorescent-labeled antibodies” Analytica ChimicaActa, (1989), 227:73-96; and Ekins, R P and F W Chu. “Multianalytemicrospot immunoassay—microanalytical ‘compact disc’ of the future”(1991), all of which are incorporated by reference.

In the case of oligonucleotide arrays, conventional in situ synthesischemistry is used. The phosphoramidite monomer unit is activatedtypically with tetrazole, for example, before the coupling reaction. Theactivated phosphoramidite is moisture sensitive and will reactimmediately with moisture in the ambient to hydrolyze thephosphoramidite. Without the method 100, 100′ of the invention, theactivated phosphoramidite will react with water vapor in ambientatmosphere, even in small amounts present in a nitrogen chamber, and bedepleted before a sufficient amount of the activated phosphoramidite hasreacted with the growing oligonucleotide chains of the array.

In still another embodiment, a method 200 is provided for fabricatingarrays of biopolymers, wherein the biopolymers are pre-synthesized andthen linked to the surface 23 of an array 20 in a desired array patternof features. Such arrays 20 of pre-synthesized biopolymers include, forexample cDNA arrays, DNA arrays or RNA arrays. In accordance with themethod 200, the surface 23 of the array substrate 20 is prepared toreceive or link to the pre-synthesized biopolymer using conventionalwell-known methods. For example, when the biopolymer is cDNA, the arraysubstrate surface 23 is coated with an aqueous solution of polylysine.Moreover, the protecting group on the pre-synthesized biopolymer isremoved (deprotection) using conventional methods, so that thebiopolymer can link to the prepared surface 23. The deprotection steprenders the pre-synthesized biopolymer water-soluble. Therefore, thedeprotected pre-synthesized biopolymer is dissolved in aqueous solutionfor deposition onto the array 20. For example, when the pre-synthesizedbiopolymer is DNA or RNA, the deprotected pre-synthesized DNA or RNA iswater-soluble and is typically dissolved in an aqueous buffer solutionfor deposition. However, the aqueous solution of pre-synthesizedbiopolymer is susceptible to evaporation, especially in smallquantities, for example, when it is deposited onto the surface of thearray for linking. Unless some precaution is taken, the aqueous solutionmay prematurely evaporate before the linking reaction occurs, or beforethe linking reaction is completed. As a result, some of thepre-synthesized biopolymer will not link to the surface 23. Therefore,the evaporation of the aqueous solution on the surface will vary theconcentration, and resulting uniformity, of deposited pre-synthesizedbiopolymers at the respective feature locations 22.

In accordance with the method 200, the feature locations or sites 22 ofthe array 20 are then covered with the NMF (step 201). The NMF may beflooded over the surface 23, as in FIGS. 2-4, or deposited as droplets31 over one or more individual sites 22, as in FIG. 6a. Alternatively,the array may be immersed into the NMF, as in FIG. 5.

Droplets of the aqueous solution of deprotected pre-synthesizedbiopolymer 28′ are deposited (step 203) on the array 20, for example, bythe pulsejet 26, as mentioned above for method 100, 100′, or with a pinor capillary, as is well known in the art. In the preferred embodiment,the pulsejet heads 26 deliver the different biopolymers 28′ to theirdesired feature locations 22 on the array 20. As described above formethod 100, 100′, the pulsejet 26 fires the droplets of biopolymer 28′into the NMF to the sites 22 or feature locations on the surface 23 ofthe array 20, where the particular biopolymer 28′ sequence is to belinked. Any of the deposition methods illustrated in FIGS. 2 to 6 a,bwill work for the method 200. One pulsejet head 26 can deliver only onesequence of pre-synthesized biopolymer 28′. After a particularpre-synthesized biopolymer 28′ sequence is delivered to all desiredlocations 22, the pulsejet head 26 is washed and reloaded with asolution of a different pre-synthesized biopolymer 28′ sequence.

In accordance with the invention, the NMF that is applied to the array20 is insoluble in aqueous solutions, such as in water and in buffersolution, such as the buffer solution used for the deprotectedpre-synthesized DNA or RNA. The NMF effectively shields each droplet 28′and impedes or delays the diffusion and subsequent evaporation of theaqueous solution, so that the linkage reaction can be completed. As aresult, the deposited pre-synthesized biopolymers 28′ have uniformconcentration throughout each feature location 22.

The NMF is then removed (step 204) from the array 20 and the array 20 iswashed with water to remove any unbound biopolymers and ancillarymaterials, preferably about 5 to 10 minutes after all of thepre-synthesized biopolymers have been deposited. If the array surface 23was treated with polylysine to prepare the surface for linkage, anadditional step of capping unreacted polylysine surface sites 22 must beperformed according to conventional methods to prevent further bindingto biopolymers, such as target sequences during an assay, which couldcompromise assay results. Whether an additional step to cap ordeactivate is needed generally depends on the biopolymers that are beinglinked and the type of linkage chemistry chosen. The additional stepsthat may be necessary are well known in the art.

The methods 100 and 200 of the invention provide a robust tool forsolving a longstanding problem in the art of making biopolymer arrayswith accuracy. The biopolymer arrays are used in assays of targetbiological materials. In order for an assay to yield accurate results,it is important that the different biopolymer features actually bepresent on the array, that they are put down accurately in the desiredor predetermined pattern and biomonomer sequence, that the biopolymersare of the correct size, and that each different feature be uniformlypopulated with the respective biopolymer. The present method 100, 200provides means to protect the sensitive reaction environment duringbiopolymer array fabrication from external environmental influences thatmight compromise the fabrication of the desired biopolymer array.Moreover, the present invention provides a means by which the potentialreactivity of an activated biomonomer or of a deprotectedpre-synthesized biopolymer with an ambient atmosphere component can bekept low. As a result, the biopolymer array fabricated in accordancewith the invention will more likely have uniformly populated biopolymersof the correct sequence and size at each array feature.

While biopolymer arrays are described above, the present inventioncontemplates that these particular moieties can readily be replaced withother moieties (such as other chemical or biochemical moieties, forexample various small molecules), where the activated component is morereactive with a component of the ambient atmosphere than is theunactivated component. Thus, wherever a reference is made tobiopolymers, this can be replaced with a reference to any such moieties.

The present invention is essentially a method of shielding sensitivebiosynthesis reactions and reactants from one or more components of theambient environment. The NMF is essentially a shield that is appliedover the biosynthesis reactions to separate the bioreactants from theambient environment while the biosynthesis reactions take place.

Thus there has been described a new method 100, 100′, 200 of fabricatingbiopolymer arrays that provide a shield for sensitive biopolymerfabrication on the array from environmental conditions which may havedetrimental effects on the accuracy of the fabrication results. Itshould be understood that the above-described embodiments are merelyillustrative of the some of the many specific embodiments that representthe principles of the present invention. Clearly, numerous otherarrangements can be readily devised by those skilled in the art withoutdeparting from the scope of the present invention.

What is claimed is:
 1. A method of fabricating a biopolymer array frombiomonomers comprising the steps of: (a) depositing a biomonomer insolution onto a substrate for linking the biomonomer to a surface of thesubstrate in an array pattern of features; (b) deprotecting the linkedbiomonomer, such that the linked biomonomer can react with subsequentbiomonomers in solution; (c) depositing a subsequent biomonomer insolution onto the features, the subsequent biomonomer being activatedfor attachment to the linked biomonomer on the array feature by anactivation reagent, the subsequent biomonomer becoming the linkedbiomonomer upon attachment; (d) applying a non-miscible fluid (NMF) overthe features on the surface of the substrate before or after any of theabove steps (a) to (c), the NMF being inert and insoluble with thebiomonomer solution, the activation reagent, the activated biomonomerand the linked biomonomer; and (e) repeating at least step (c) until thebiopolymer is synthesized at each feature.
 2. The method of claim 1,wherein the biopolymer array comprises multiple different array featuresand steps (a) through (d) are repeated at each of multiple differentfeatures on the same substrate, to produce the array of multipledifferent biopolymers.
 3. The method of claim 1, wherein the step ofapplying (d) the NMF comprises the step of depositing an amount of NMFso as to cover an area greater than the area of each feature.
 4. Themethod of claim 3, wherein the step of depositing an amount of NMFcomprises the step of depositing a droplet of NMF on each feature thatis large enough to completely cover the feature, and wherein the steps(a) and (c) of depositing the biomonomers comprise the steps of: loadingthe biomonomer in solution into a pulsejet of a deposition system;positioning the pulsejet over the NMF droplet on the surface of thearray; ejecting the biomonomer from the pulsejet into the NMF dropletand to the feature; moving the pulse jet to a next feature; andrepeating the steps of positioning, ejecting and moving for eachapplicable feature location until the biomonomer is deposited on allapplicable features.
 5. The method of claim 3, wherein the step ofdepositing an amount of NMF comprises the step of covering the entirearray surface and completely covering the features with the NMF.
 6. Themethod of claim 5, wherein the steps (a) and (c) of depositing thebiomonomers comprise the steps of: loading the biomonomer in solutioninto a pulsejet of a deposition system; immersing the pulsejet into theNMF over a feature on the surface of the array; ejecting the biomonomerfrom the pulsejet to the feature; moving the immersed pulse jet to anext applicable feature location and ejecting the biomonomer to theapplicable feature; and repeating the step of moving and ejecting untilthe biomonomer is deposited on all applicable features.
 7. The method ofclaim 5, wherein the steps (a) and (c) of depositing the biomonomerscomprise the steps of: loading the biomonomer in solution into apulsejet of a deposition system; immersing the pulsejet into the NMFover a feature on the surface of the array; ejecting the biomonomer fromthe pulsejet to the feature; removing the pulse jet from the NMF andmoving the pulse jet to a next applicable feature location; andrepeating the steps of immersing, ejecting and removing over eachapplicable synthesis site until the biomonomer is deposited on allapplicable features.
 8. The method of claim 5, wherein the steps (a) and(c) of depositing the biomonomers comprise the steps of: loading thebiomonomer in solution into a pulsejet of a deposition system;positioning the pulsejet above the NMF over a feature location on thesurface of the array; ejecting the biomonomer from the pulsejet and intothe NMF to the feature; moving the pulse jet to a next applicablefeature location; and repeating the steps of positioning, ejecting andmoving over each applicable feature location until the biomonomer isdeposited on all applicable features.
 9. The method of claim 5, whereinthe steps (a) and (c) of depositing the biomonomers comprise the stepsof: loading the biomonomer in solution into a pulsejet of a depositionsystem; immersing the array surface into the NMF and aligning a featureon the array surface with the pulsejet; ejecting the biomonomer from thepulsejet to the feature; moving the array such that a next applicablefeature location is aligned with the pulse jet; and repeating the stepsof ejecting and moving for each applicable feature location until thebiomonomer is deposited on all applicable features.
 10. The method ofclaim 1, wherein the NMF has a density that is different from thedensity of the biomonomer solution.
 11. The method of claim 10, whereinthe NMF has a lower density than the density of the biomonomer solution.12. The method of claim 10, wherein the NMF has a higher density thanthe density of the biomonomer solution.
 13. The method of claim 1further comprising the steps of: (d′) deactivating unreacted activationreagent, the deactivation reagent having a density that is differentfrom the NMF; and (d″) removing the NMF, reagents, and unreactedbiomonomer solution from the array surface before the step (e).
 14. Themethod of claim 13, wherein the step of deactivating (d′) compriseshydrolyzing the activation reagent and unreacted biomonomer.
 15. Themethod of claim 13, wherein the biopolymer is selected from a groupconsisting of an oligonucleotide and polynucleotide; the biomonomers areselected from a group consisting of phosphoramidites, phosphites andH-phosphonates; the solution comprises a solvent selected from a groupconsisting of acetonitrile, propylene carbonate and adiponitrile; theactivation reagent is selected from a group consisting of tetrazole,ethylthiotetrazole, dicyanoimidazole and benzimidazolium triflate; thedeactivation reagent is selected from a group consisting of methanol,water, an alkyl alcohol, a halogenalkylalcohol, and trichloroethanol;and wherein the NMF is selected from a group consisting of heptane,octane, nonane, decane, undecane, dodecane, tridecane, tetradecane,pentadecane, hexadecane, heptadecane, cycloheptane, cyclooctane,cyclononane, and cyclodecane.
 16. The method of claim 1, where the NMFis applied after the biomonomer is linked to the surface of thesubstrate, but before the subsequent biomonomer and activation reagentare deposited.
 17. The method of claim 1, where the NMF is appliedbefore the biomonomer is deposited and linked to the surface of thesubstrate.
 18. In a method of fabricating a biopolymer array frombiomonomers synthesized on the array by depositing and linking abiomonomer from a biomonomer solution to a feature location on a surfaceof the array, and depositing a subsequent biomonomer in solution ontothe feature location that is suitably activated with an activationreagent to couple with the linked biomonomer, wherein the above stepsare repeated to form a biopolymer sequence at multiple feature locationson the array, the improvement comprising the step of: applying anon-miscible fluid (NMF) over the feature locations on the surface ofthe array, the NMF being inert and insoluble with the biomonomersolution and activation reagent.
 19. The method of claim 18, where theNMF is applied after the biomonomer is linked to the surface of thearray, but before the subsequent biomonomer and activation reagent aredeposited.
 20. The method of claim 18, where the NMF is applied beforethe biomonomer is deposited and linked to the surface of the array. 21.The method of claim 18, wherein the step of applying a NMF comprises thestep of depositing an amount of NMF so as to cover an area greater thanthe area of each feature location.
 22. The method of claim 21, whereinthe step of depositing an amount of NMF comprises the step of coveringthe entire array surface and feature locations with the NMF.
 23. Themethod of claim 18, wherein the NMF has a density that is different fromthe density of the biomonomer solution.
 24. The method of claim 23,wherein the NMF has a lower density than the density of the biomonomersolution.
 25. The method of claim 23, wherein the NMF has a higherdensity than the density of the biomonomer solution.
 26. The method ofclaim 18 further comprising the steps of: deactivating unreactedactivation reagent with a deactivation reagent, the deactivation reagenthaving a density that is different from the NMF, the NMF being inert andinsoluble in the deactivation reagent; and removing the NMF, reagentsand unreacted biomonomer solution from the array surface before anotherbiomonomer is deposited onto the array.
 27. The method of claim 26,wherein the step of deactivating comprises hydrolyzing the activationreagent and unreacted biomonomer.
 28. The method of claim 26, whereinthe biopolymer is selected from a group consisting of an oligonucleotideand polynucleotide; the biomonomers are selected from a group consistingof phosphoramidites, phosphites and H-phosphonates; the solutioncomprises a solvent selected from a group consisting of acetonitrile,propylene carbonate and adiponitrile; the activation reagent is selectedfrom a group consisting of tetrazole, ethylthiotetrazole,dicyanoimidazole and benzimidazolium triflate; the deactivation reagentis selected from a group consisting of methanol, water, an alkylalcohol, a halogenalkylalcohol, and trichloroethanol; and wherein theNMF is selected from a group consisting of heptane, octane, nonane,decane, undecane, dodecane, tridecane, tetradecane, pentadecane,hexadecane, heptadecane, cycloheptane, cyclooctane, cyclononane, andcyclodecane.